Contamination with Depleted or Enriched Uranium Differently Affects Steroidogenesis Metabolism in Rat

Abstract

Uranium is a naturally occurring heavy metal found in the Earth’s crust. It is an alpha-emitter radioactive element from the actinide group that presents both radiotoxicant and chemotoxicant properties. Some studies revealed that uranium could affect the reproductive system. To distinguish chemical versus radiological effects of uranium on the metabolism of the steroids in the testis, rats were contaminated via their drinking water with depleted or enriched uranium. Animals were exposed to radionuclides for 9 months at a dose of 40 mg/L (560 Bq/L for depleted uranium, 1680 Bq/L for enriched uranium). Whereas depleted uranium did not seem to significantly affect the production of testicular steroid hormones in rats, enriched uranium significantly increased the level of circulating testosterone by 2.5-fold. Enriched uranium contamination led to significant increases in the mRNA levels of StAR (Steroidogenic Acute Regulatory protein; 3-fold, p = .001), cyp11a1 (cytochrome P45011a1; 2.2-fold, p < .001), cyp17a1 (cytochrome P45017a1; 2.5-fold, p = .014), cyp19a1 (cytochrome P45019a1; 2.3-fold, p = .021), and 5α-R1 (5α reductase type 1; 2.0-fold, p = .02), whereas depleted uranium contamination induces no changes in the expression of these genes. Moreover, expression levels of the nuclear receptors LXR (Liver X Receptor) and SF-1 (Steroidogenic Factor 1), as well as the transcription factor GATA-4, were modified following enriched uranium contamination. Altogether, these results show for the first time a differential effect among depleted or enriched uranium contamination on testicular steroidogenesis. It appears that the deleterious effects of uranium are mainly due to the radiological activity of the compound.

Uranium is the heaviest naturally occurring element found in the Earth’s crust. It is an alpha-emitter radioactive element from the actinide group that present both radiotoxicant and chemotoxicant properties. This radionuclide is a mixture of three radioactive isotopes: ²³⁸U, ²³⁵U, and ²³⁴U. In naturally occurring uranium, ²³⁵U only accounts for 0.72% of the isotopic composition (Craft et al. 2004). Therefore, techniques have been developed in which uranium is chemically enriched, increasing the percentage of ²³⁵U to 2%–4%. The by-product of the enrichment process, commonly called depleted uranium (DU), has decreased levels of ²³⁵U, and is two time less radioactive than natural uranium (Craft et al. 2004). However, besides natural presence in ground, human applications release uranium through mill tailings, nuclear industry, and military army. Thus the population could be highly exposed to uranium via their drinking water or alimentation (Craft et al. 2004).

Chemically, uranium causes toxicity as a heavy metal, and it has been shown that the major health concern from DU is toxicological rather than radiological (Sztajnkrycer and Otten 2004). Some studies analyzed separately, the effect of depleted or enriched uranium (EU) on several biological systems. Indeed it has been demonstrated that chronic ingestion of DU affects the expression of drug-metabolizing cytochrome P450 (CYP) enzymes, as well as the nuclear receptors that regulate these enzymes (Gueguen et al. 2007; Souidi et al. 2005). Besides, uranium has been shown to modulate activity of CYP involved in lung and liver testosterone catabolism (Pasanen et al. 1995). Hormonal changes have also been shown after long-term exposure to low doses of uranium. Indeed, Namibian uranium miners present a significantly reduce testosterone levels, probably reflecting damage to gonadal endocrine system (Zaire et al. 1997). However, no significant effects were observed for steroid hormones concentrations in Gulf war veterans with retained fragments of DU shrapnel (McDiarmid et al. 2000). Effects of contamination on reproduction have also been studied using rats under normalized conditions. Orally contamination of male and/or female rats with high dose of depleted uranium (10 to 40 mg/kg/day) induces a significant decrease in the pregnancy rate of treated rats, and shows some abnormal morphological forms and sperm parameters modifications, together with accumulation of depleted uranium in the testis (Linares et al. 2005). Likewise, high dose of enriched uranium (EU) contamination has been linked to spermatozoa morphologic modifications, and chromosome aberration in spermatogonia (Zhu, Hu, and Lun 1994).

These studies, however, do not distinguish the chemical versus the radiological effects of uranium. Besides, uranium contamination also affects vitamin D plasma level, which decreases after DU chronic ingestion, whereas no change is observed following EU contamination (Tissandie et al. 2007, 2008), even though expression of vitamin D–responsive genes involved in renal calcium transport decreases following both DU and EU contamination. Moreover, rats exposed to 4% enriched uranium for 1.5 months through drinking water present an increase of about 38% of the amount of paradoxical sleep, a reduction of their spatial working memory capacities, and an increase in their anxiety (Houpert et al. 2005). Similar exposure to DU did not induce these effects. Altogether, these results show that sensibility to chemical or radiological properties of uranium differs among organs (e.g., kidney and the central nervous system [CNS]).

To date, few studies have been conducted regarding the reproductive dysfunctions following inner contamination by uranium in animal models, and particularly distinguishing between chemical or radiological effects. In this paper, contamination procedures were realized using low doses of DU or EU (40 mg/L, 1 mg/rat/day), during 9 months to investigate the contamination effects on the testicular endocrinology in rats. This dose represent double highest concentration find in some wells of Finland (Juntunen 1991; Salonen 1994). Blood testosterone and estradiol levels were measured, as well as gene expression of enzymes (cytochrome P450, hydroxysteroid dehydrogenase), transporters (StAR), and nuclear receptors involved in this metabolism.

MATERIALS AND METHODS

Chemicals and Materials

Depleted uranyl nitrate hexahydrate (98.74% ²³⁸U, 0.26% ²³⁵U, 0.001% ²³⁴U) and enriched uranium nitrate (95.74% ²³⁸U, 4.24% ²³⁵U, 0.02% ²³⁴U) were obtained from AREVA-NC (France).

Animals

Sprague-Dawley male rats (Charles River, L’Arbresle, France), weighing 250 g each, were divided into four groups of 10 rats each: two control groups and two experimental groups. The rats were housed in pairs, with a 12-h light/12-h dark cycle (light on 08:00 h/20:00 h) and a temperature of 22°C ± 1°C. Water and food were delivered ad libitum. The main biochemical parameters of these animals were published elsewhere (Tissandie et al. 2008; Souidi et al. 2005). All experimental procedures were approved by the Animal Care Committee of the Institute of Radioprotection and Nuclear Safety and complied with French regulations for animal experimentation (Ministry of Agriculture Act No. 87-848, October 19, 1987, modified May 29, 2001). During the DU contamination experiment, rats within the control group chosen to be included in the hormone analysis died. We decided thus to remove two animals in the contaminated group.

Contamination Procedures

Rats were exposed to DU (specific activity 1.4 × 10⁴ Bq/g) or EU (specific activity 4.2 × 10⁴ Bq/g) at a dose of 40 mg/L (560 Bq/L for DU, 1680 Bq/L for EU), in their drinking water for 9 months. Because the contaminations procedures were not performed simultaneously, each exposed group had its own control group.

Real-Time PCR on Testicular Tissues

Testicular total RNA was prepared with the RNeasy total RNA isolation Kit (Qiagen, Courtaboeuf, France) according to the manufacturer’s instructions. The cDNA was produced from 1 μg of total RNA by reverse transcription with BD Sprint PowerScript PrePrimed 96 plate (BD Biosciences clontech, Erembodegem, Belgium). Real-time polymerase chain reaction (PCR) was performed on an Abi Prism 7900 Sequence detection System (Applied Biosystems, Courtaboeuf, France) using 10 ng of template DNA for each reaction. Samples were normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) (Ropenga et al. 2004). Analysis of relative gene expression data were realized using the ΔΔC _t method. Sequences for the primers are indicated in Table 1.

Hormonal Assays

Hormones (testosterone, 17β-estradiol) were analyzed by enzyme-linked immunosorbent assay (ELISA) on serum or plasma, using kits from Abcys (Paris, France), according to the manufacturer’s instructions. Briefly, hormone in the sample competes with enzyme-labeled antigen for antibody binding. Samples were dispensed into coated plates and then the enzyme conjugate was added. During the incubation, competition for binding sites took place. The bound conjugated enzyme was then detected. The intensity of the detection was inversely proportional to the amount of hormone in the sample.

Statistical Analysis

Results are reported as mean ± SE. Statistical analyses were performed with Student’s ttest, or Mann-Whitney test. The Mann-Whitney test was used for nonparametric values. Differences were considered significant when p < .05.

RESULTS

Chronic Contamination with EU But Not DU Affects Sex Steroid Status in Male Rats

The groups of rats ingested the radionuclides through the drinking water for 9 months, with double the highest dose naturally found in some wells. These protocols were thus considered as a chronic contamination, as previously described (Souidi et al. 2005). DU and EU contaminations did not affect food intake, weight gain, or animal general health status. Besides, the macroscopic appearance of testes, liver, kidney, lungs, intestines, or brain did not show any sign of toxicity, and did not differ from that of the control rats (data not shown).

To analyze the effects of these chronic contaminations on testis functioning, the main sex steroids were measured in the blood. Whereas DU exposure did not seem to significantly affect the production of testosterone and 17β-estradiol in rats (Table 2), EU exposure significantly increased the level of circulating testosterone by 2.5-fold (0.26 ± 0.04 ng/ml for the control group versus 0.67 ± 0.09 for the EU-contaminated group; p < .05). The variations of the hormone levels observed among the control groups (control of the DU experiment and control of the EU experiment) were explained by the use of plasma for UE analyses versus serum for DU analyses.

EU-Treated Rats Have Increased Expression of Genes Involved in the Steroidogenesis

Because EU induced variations of the circulating sex steroid, we aimed to investigate the effects of various chronic contaminations on the levels of the messengers encoding the proteins regulating the steroid synthesis. We analyzed by quantitative PCR (qPCR) the messenger levels of steroid acute regulatory protein (StAR), responsible for the shuttling of the cholesterol from the outer to the inner membrane of the mitochondria; the cytochrome P450 side chain cleavage enzyme (cyp11a1), which forms pregnenolone from cholesterol; cytochrome P450 17α-hydroxylase (cyp17a1); 3β- and 17β-hydroxysteroid dehydrogenases (3β-HSD1 and 17β-HSD3); 5α-reductase (5α - R1), which synthesizes dihydrotestosterone from testosterone; and aromatase (cyp19a1), which produces 17β-estradiol.

As expected, because no hormonal change occurred, no significant modification of the expression levels of the genes was observed in rats contaminated with DU (Figure 1). EU contamination, however, led to significant increases in the levels of StAR (3-fold, p = .001), cyp11a1 (2.2-fold, p < .001), cyp17a1 (2.5-fold, p = .014), which correlates with the higher levels of testosterone (2.5-fold compare to the control) in these contaminated animals. Besides, levels of cyp19a1 (2.3-fold, p = .021) and 5α-R1 (2-fold, p = .02) were also increased by the contamination with EU. Moreover, levels of apoD, encoding apolipoprotein D involved in the transport of cholesterol as well as steroids in the testis, was not affected (data not shown).

EU Affects the Levels of Transcription Factors Involved in the Control of Steroidogenesis and Male Fertility

In order to determine the molecular mechanisms leading to the variations of the steroidogenic genes, expression levels of the transcription factors regulating the expression of these genes were measured by qPCR Steroidogenic factor 1 (SF-1), which positively regulates StAR, cyp11a1, cyp17a1, and cyp19a1, and its opposite counterpart DAX-1 (dosage-sensitive adrena congenita hypoplasia on the X-chromosome, locus 1) were analyzed. Likewise, farnesoid X-receptor (FXR) and small heterodimeric partner (SHP), both shown to down-regulate steroidogenesis; the liver X-receptors α and β (LXRα, LXRβ), which activate testicular steroidogenesis in mouse and controls its fertility, respectively; and receptor for 9-cis retinoic acid α (RXRα), which heterodimerizes with the above factors; were studied. The messenger encoding the factor GATA-4, which regulates expression of SF-1, was also measured. As previously shown for the other parameters, DU treatment did not induce any variation (Figure 2). Conversely, EU contamination significantly increased the levels of GATA-4 (87%, p = .033) and SF-1 (64%, p = .031), both involved in the cascade of regulation of steroidogenic genes, compared to the untreated group. An increase of LXRβ (2-fold, p < .02) and its heterodimeric partner RXRα (2.8-fold, p = .008) was also found. The puzzling fact is that the steroidogenesis inhibitor SHP is also significantly increased (54%, p = .032). Note that the expression level of genes encoding the peroxisome proliferator–activated receptors (PPARs) α and γwere not affected by any of the contaminations (data not shown).

DISCUSSION

The major health concern from DU is toxicological rather than radiological (Sztajnkrycer and Otten 2004). Discrimination between radiological and chemical effects of uranium can be realized by comparing effects obtained with depleted or enriched uranium. For example, this discrimination has been studied on vitamin D metabolism, as well as on the CNS. Differential effects of DU or EU on these two systems have been demonstrated.

Vitamin D plasma level decreases after chronic DU contamination, but not EU contamination (Tissandie et al. 2007, 2008), whereas expression of vitamin D target genes involved in renal calcium transport decrease following both exposure. Likewise, rats exposed to EU through drinking water present an increase of the amount of paradoxical sleep, a reduction of their spatial working memory capacities, and an increase in their anxiety, whereas DU has no effect (Houpert et al. 2005).

Some studies have been carried out on the effects of natural and depleted uranium on rodent reproductive system. Mouse studies investigating chronic exposure to naturally occurring uranium demonstrated a significant but not dose-related decline in male fertility, without alteration of the testicular function (Domingo 2001; Llobet et al. 1991). However, studies in male rats have suggested a direct gonadotoxic effect of DU, resulting in testicular atrophy and germ cell depletion (Domingo 2001).

In our study, we investigated the role of a chronic and long-term contamination by DU and EU on the sex steroidogenesis in male rats. Even though the general health status of the animals was not affected, nor was food intake or weight gain, our results pointed out a differential effect of this contamination depending on the enrichment level of uranium. Indeed, although DU had no significant effect, we showed for the first time an effect of EU on the testosterone level in blood, paralleled with significant changes in the amount of mRNA encoding the steroidogenic proteins, as well as those of the transcription factors regulating them. As shown on Figure 3, EU contamination seems to modify two pathways of steroidogenesis regulation: induction of expression of LXRα/RXRα, which could act on StAR, and GATA-4, which triggers SF-1 expression, which in turn increases the levels of StAR, cyp11a1, cyp17a1, and cyp19a1. Altogether, EU contamination has a clear-cut effect on steroidogenesis in testis. It seems that the primum movens of this increase is the alteration of the transcriptional regulation of GATA-4 (Figure 3). Interestingly, the levels of 5α-R1 were also increased. This point has been evoked to explain the paradoxical increase of testosterone levels and gene expression level of aromatase without modification of the estrogens levels. The elevated testosterone level is not in agreement with observation of a reduced testosterone level among Namibian uranium miners (Zaire et al. 1997). This difference could be explained by the route of contamination (inhalation for miners versus ingestion for rats), or the nature of uranium (natural versus enriched).

As observed, DU contamination did not seem sufficient to induce a hormonal perturbation of the testis because chronic ingestion of this heavy metal with a low radiologic activity (560 Bq/L) had poor effect on testicular steroidogenesis of young adult rats. The reason why testis physiology was not affected by DU is still under investigation. Altogether, these results suggest that the radiologic activity of uranium, more than the toxic effect of the heavy metal, affects the testicular steroidogenesis. This radiologic versus chemical sensibility seems to be tissue specific.

In conclusion, our results show for the first time a differential effect among the various contaminating radionuclides on the testicular steroidogenesis. It appears that the deleterious effects are mainly due to the radiological activity of the compounds. Whether a chronic contamination could also affect the pituitary-gonadal axis as well as testis structure and reproduction is the future challenge of these studies. Even though biochemistry experiments have not been performed yet, the original fact that uranium alters testicular endocrinology will allow developing of new concepts about chronic contaminations by uranium and male reproductive defects in human, and will open a new filed of investigation in understanding how uranium could affect transcription levels.

Abbreviations: CYP, cytochrome P450; DU, depleted uranium; EU, enriched uranium; hprt, hypoxanthine-guanine phosphoribosyltransferase; FXR, farnesoid X receptor; LXR, liver X receptor; RXR, retinoid X receptor, SHP, small heterodimeric partner; StAR, steroidogenic acute regulatory protein; 3β-HSD, 3beta hydroxysteroid dehydrogenase; 17β-HSD, 17beta hydroxysteroid dehydrogenase; SF-1, steroidogenic factor 1.